Strain background can strongly influence the outcome of toxicity tests in animals. Time and cost constraints preclude in vivo approaches to longstanding concerns about the lack of genetic diversity in animal models. An in vitro platform is vastly more efficient for understanding the role of genetic background in toxicology testing, and sacrifices no animals. In this project we propose to build a large panel of genetically diverse ES cells from the Diversity Outcross line of mice. The DO mice are an advanced intercross of the 8 founder strains used for the Collaborative Cross. The DO strains capture almost all the sequence variants in laboratory strains, and harbor hundreds to thousands more recombinational breakpoints than F2 mice. They ideally suited for facilitate complex trait mapping. ES cells offer tremendous flexibility and is also ideal for an in vitro genetics platform. DO ES cell lines immortalize DO genomes, creating a renewable resource. In principle, ES cells can provide access to almost any cell type by directed differentiation. In this way, the DO ES panel is extremely versatile as a permanent, renewable resource. In Phase I, we will establish feasibility by deriving 90 independent, validated DO ES lines and estimate the genetic component of cytotoxicity observed for 15 model toxicant compounds in the Phase I panel. In Phase II we will complete the 600 ES line panel, identify compounds with a significant genetic component to cytotoxic response using a 1400 reference compound set, and map the loci mediating that genetic component in the identified compounds. PUBLIC HEALTH RELEVANCE: There is a longstanding interest in how genetics impacts toxicology, but cost effective, highly scalable tools to investigate this relationship were not available. This project will improve human health and reduce the risk of environmental toxicants by developing a high throughput in vitro platform for investigating the genetic basis of variable response to toxicant exposure.